In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56ac) is found in new histones deposited behind DNA replication forks and is needed for DNA damage survival. Genome-wide removal of H3K56ac by the deacetylases Hst3 and Hst4 occurs during G2 and/or M phase. H3K56ac is a double-edged sword. Lack of H3K56ac results in DNA damage sensitivity. In contrast, overabundance of H3K56ac in hst3Δ hst4Δ mutants gives rise to even more severe and wide-ranging phenotypes, namely thermosensitivity, genotoxic agent hypersensitivity, genome instability and short replicative lifespan. The deacetylases Hst3 and Hst4 are tightly controlled during the cell cycle such that H3K56ac can contribute to the DNA damage response during each passage through S phase, while avoiding abnormal conditions where H3K56 remains hyperacetylated. In this thesis, we identified the molecular machinery that promotes Hst3 degradation. Moreover, we explored why failure to deacetylate H3K56 gives rise to the phenotypes of hst3Δ hst4Δ cells.
In chapter 2, we showed that degradation of Hst3 can be completed prior to anaphase. This suggests that removal of H3K56ac occurs during a short time window between completion of S phase and metaphase. In addition, we found that Hst3 is phosphorylated at two cyclin-dependent kinase 1 (Cdk1) sites and demonstrated that these phosphorylation events promote degradation of Hst3 in vivo. Moreover, we demonstrated that the ubiquitin-conjugating enzyme Cdc34 and the SCFCdc4 ubiquitin ligase are required for degradation of Hst3. Lastly, we showed that phosphorylation of Hst3 by the mitotic kinase Clb2-Cdk1 can directly drive its ubiquitylation by SCFCdc4 in vitro.
In chapter 3, we investigated the molecular mechanisms that underlie the severe sensitivity of hst3Δ hst4Δ cells to DNA damaging agents. We established that the aberrant presence of H3K56ac in front of DNA replication forks causes hst3Δ hst4Δ cells to lose viability after a single passage through S phase in the presence of methyl methanesulfonate (MMS). We also found that, although hst3Δ hst4Δ cells show normal activation of the DNA damage checkpoint, these mutants fail to complete DNA replication and inactivate the checkpoint long after MMS removal. Collectively, our results suggest that MMS-induced DNA lesions cause a severe loss of viability in hst3Δ hst4Δ cells because the mutant cells fail to complete DNA replication after MMS removal.
In the second part of chapter 3, we employed a genetic approach to identify novel mechanisms for suppression of two pronounced phenotypes of hst3Δ hst4Δ mutants. We found that deletion of several genes involved in creating boundaries between heterochromatic and euchromatic regions alleviates the phenotypes of hst3Δ hst4Δ mutants without reducing H3K56 hyperacetylation. Our results also indicate that the highly abundant histone H4 lysine 16 acetylation (H4K16ac) is deleterious to hst3Δ hst4Δ mutants, suggesting an intriguing and hitherto undiscovered genetic link between H3K56ac and H4K16ac. We identified a group of spontaneous suppressors that exhibited undetectable levels of H3K56ac, but the majority did not show obvious decreases in H3K56ac or H4K16ac compared to the levels observed in hst3Δ hst4Δ cells. Further characterization of these suppressors might unravel additional genetic or epigenetic mechanisms that circumvent the catastrophic consequences of H3K56 hyperacetylation in hst3Δ hst4Δ cells.
In summary, this thesis describes the molecular machinery that triggers destruction of the main H3K56 deacetylase Hst3 during a period of time delineated by the end of S phase and metaphase. Our findings also explain why degradation of Hst3 always precedes the onset of S phase when H3K56ac needs to accumulate behind DNA replication forks in order to act as defense mechanism against DNA damage. In addition, we uncover several novel suppressors that bypass the role of Hst3 and Hst4 in DNA damage resistance. Several suppressors reveal an unexpected genetic link between two abundant forms of histone acetylation, namely H3K56ac and H4K16ac.